Pyruvate anaplerosis is a targetable vulnerability in persistent leukaemic stem cells

Deregulated oxidative metabolism is a hallmark of leukaemia. While tyrosine kinase inhibitors (TKIs) such as imatinib have increased survival of chronic myeloid leukaemia (CML) patients, they fail to eradicate disease-initiating leukemic stem cells (LSCs). Whether TKI-treated CML LSCs remain metabolically deregulated is unknown. Using clinically and physiologically relevant assays, we generate multi-omics datasets that offer unique insight into metabolic adaptation and nutrient fate in patient-derived CML LSCs. We demonstrate that LSCs have increased pyruvate anaplerosis, mediated by increased mitochondrial pyruvate carrier 1/2 (MPC1/2) levels and pyruvate carboxylase (PC) activity, in comparison to normal counterparts. While imatinib reverses BCR::ABL1-mediated LSC metabolic reprogramming, stable isotope-assisted metabolomics reveals that deregulated pyruvate anaplerosis is not affected by imatinib. Encouragingly, genetic ablation of pyruvate anaplerosis sensitises CML cells to imatinib. Finally, we demonstrate that MSDC-0160, a clinical orally-available MPC1/2 inhibitor, inhibits pyruvate anaplerosis and targets imatinib-resistant CML LSCs in robust pre-clinical CML models. Collectively these results highlight pyruvate anaplerosis as a persistent and therapeutically targetable vulnerability in imatinib-treated CML patient-derived samples.


Reporting on sex and gender
Reporting on race, ethnicity, or other socially relevant groupings 0.940), was used to trim bases with quality scores of less than 20. Prior to and after this pre-processing fastqc (version 0.11.2) was run to ascertain sequence quality, alongside the efficacy of the pre-processing steps. Trimmed reads were indexed and aligned using Hisat2 (version 2.1.0). Hisat2 indexes (GRCh38 genome_tran) were obtained from the John Hopkins Center for Computational Biology, 2020. Samtools (version 0.1.19044428cd) view was used to convert the resulting .sam to .bam files, whilst samtools sort was used to sort the .bam files. Assembly was achieved through the use of stringtie (John Hopkins Center for Computational Biology, 2020), with output .gtf files converted to count matrices using the python script prepDE.py (stringtie version 1.3.3b.Linux_x86_64). Reads were assembled using an annotated reference human genome (GRCh38.p13), obtained from GENCODE (GENECODE, 2020). DESeq2 (version 1.26.0) was used to generate results sets from the gene and transcript count matrices. G genes with read counts too low to allow for the calculation of p and adjusted p-values (padj: Benjamini-Hochberg) were removed from the data sets leaving gene and transcript counts of sizes 16,069 and 45,218 respectively. Microarray datasets were analysed using Limma (version 3.34.9) GSEA (version 4.1) was conducted on pre-ranked lists (ranked by pi score calculated by multiplying LOG fold change by -LOG (corrected pvalue)).
Statistical analysis was performed using R Studio version 1.1.4., MetabonAnalyst 5.0, or Graphpad Prism 9.1. LC-MS peak intensities were Rlog-transformed and mean-centered to ensure normality of data. For in vitro and in vivo data, normality was tested with non-parametric tests being used on non-parametric data.A multivariable Cox proportional hazards model was fitted to TCGA data in R software. Two datasets were analysed, one including and the other excluding the FAB M3 subtype.
To simplify the model, a backward stepwise model selection procedure was applied to the complex Cox survival model, which originally included age, sex, FLT3_ITD, protocol, transplant_type, PC, FAB, and cytogenetic_risk predictors (full model). The reduced model (reduced model) was obtained by retaining age, FLT3_ITD, protocol, transplant_type, PC, and cytogenetic_risk predictors, while dropping the interaction term between PC and FAB, from the original model.
The resulting models can be represented as: Full model: proportional hazard~age + sex + FLT3_ITD + protocol + transplant_type + PC * (FAB + cytogenetic_risk) Reduced model: proportional hazard~age + FLT3_ITD + protocol + transplant_type + PC * cytogenetic_risk Survival plot was generated to illustrate the effect of PC expression on overall survival in patients with high-risk cytogenetics, stratified by low or high (20th and 80th percentiles) PC expression levels. The 95% confidence intervals were represented by the boundaries of mean ± 1.96 * standard deviation.
The expression profiling RNA-seq data generated in this study have been deposited in public Gene Expression Omnibus (GEO) database under accession code GSE216837 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE216837]. The publicly available datasets used in this study are available in the EMBL-EBI database under accession code E-MTAB-2581 [https://www.ebi.ac.uk/biostudies/ arrayexpress/studies/E-MTAB-2581]. The remaining data are available within the Article, Supplementary Information or Source Data file.
Standard software packages LCMS data for analysis of patient samples is in source files. Raw LCMS files generated in this study are available upon request to the corresponding author immediately upon approval of biobanks ethical approval panel and access will not be time-limited. The LCMS samples will be maintained long-term (> 10 years) and raw LCMS files will be maintained indefinitely (>10 years on institutes network drive, Redundant Array of Independent Disks (RAID)). Additional information concerning human samples can be obtained from the corresponding author. Source data are provided with this paper.
These results are not sex specific. While more male patient samples were used (17) than female (8) this was due to the amount of cells available for each patient in biobank.
This data was not collected.

April 2023
Population characteristics

Recruitment
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All studies must disclose on these points even when the disclosure is negative.  (2017)) to ensure sufficient data-points without using excessive numbers of patient samples. For in vivo experiment, prior power calculations were used to estimate the number of mice per experimental arm, factoring in the efficacy of treatment observed from the in vitro data.

No relevant data was excluded in the analysis
The number of replicates performed is provided manuscript,. This was 3-4 independent replicates for cell line experiments with exact numbers provided in figure legends. Separate patient samples are counted as biological replicates. All experiments for the data and source data is presented, and irrespective of statistical differences be groups, were repeated independently as stated in manuscript.
All experimental mice were randomized (by cage) to the various experimental cohorts prior to treatment. LCMS samples were randomized during data acquisition. No other randomization was performed Researchers were not blinded as it was essential to know which treatments were required for each arm of experiments in case of adverse reaction to single agents or combination treatment or it was not possible due to analysis (e.g. CFCs needed to be counted from 6 well plate). Note that full information on the approval of the study protocol must also be provided in the manuscript.

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Study protocol
AMPK: validated in various cell lines by manufacturer p-AMPK: validated by manufacturer using AICAR treatment or starvation to induce increase p-CRKL: validated by manufacturer using phosphatase treated K562 cell lysate and here using imatinib on CMLsamples ( Figure S2O) CDK: validated by manufacturer using siRNA knock down combined with immunofluroscent analysis. h3: validated by manufacturer using sodium butyrate (inhibits deacetlyation). PC: validated by manufacturer (IP) as well as knock out generated in this study ( Figure 5A and Figure S5C). annexin V-FITC: validated in cell lines by manufacturer as reactive to all mammalian species and authors using complementary measurement of cell death (7-AAD) muCD45-APC-CY7: validated by manufacturer on mouse splenic leukocytes alongside isotype control huCD45-FITC: validated by manufacturer on human peripheral blood lymphocytes alongside isotype control huCD34-APC: validated by manufacturer on human peripheral blood stem cells huCD38-PerCP: Validated by manufacturer on human peripheral blood lymphocytes alongside isotype control K562, KCL22 and HEK-293FT cell lines were originally purchased from DSMZ and cultured following the manufacturer's instructions.
K562, KCL22 and HEK-293FT cell lines were authenticated using short-tandem repeat (STR) profiling  (2021)). Bone marrow was isolated from the hips and hind limbs through centrifugation as described in sample preparation section.
No wild animals were used in the study.
Only female mice were used for all PDX experiments as engraftment is low and variable in male mice No field collected samples were used in the study.
All animal experiments were conducted in accordance with the regulations outlined by the Animals (Scientific Procedures) Act. All experiments were conducted using personal licence number IE2DD924E and project licence PP2518370.